DNA damage-independent monoubiquitylation of hRFC2 D228A.
A, schematic diagram and tertiary model of human (Hs) RFC2
showing the location of Asp-228 and the sequences of the surrounding regions.
Corresponding sequences for S. cerevisiae (Sc) RFC2(p40) and
mouse (Mm) RFC2 homologues are also shown. The conserved Sensor 2
helix is represented by a box, and the location of the conserved SRC
motif is indicated by an arrow. Asp-228 of hRFC2, shown in
red, corresponds to S. cerevisiae Asp-201, which shows
synthetic lethality with mutation in Rpa-1(rfa1-Y29H). There are
seven conserved RFC boxes numbered consecutively from the N terminus
to C terminus. B, 293A cells were transfected with expression vectors
encoding wild-type (lanes 2 and 6), D228N (lanes 3
and 7), or D228A (lanes 4 and 8) forms of hRFC2-HA.
24 h after transfection cells were harvested and separated into chromatin
(lanes 5–8) and soluble fractions (lanes 1–4)
and then immunoblotted with anti-RFC2 or anti-PCNA antibody. The
arrowheads indicate the position of molecular mass markers (kDa).
C, Western blot of lysates from HCT116 cells (WILD) or
RAD18-deficient HCT116 cells (RAD18-/-). HCT116
cells transfected with pCAGGS·hRFC2(Asp-228) were treated with 0.85
mm MMS for 8 h. Chromatin fractions from the resulting cells were
analyzed by Western blotting with anti-RFC2 antibody. The arrowheads
indicate the position of molecular mass markers (kDa). DMSO,
Me2SO.